Interleukin-6 is a multi-functional cytokine (Kishimoto, T. and T. Hirano, Ann. Rev. Immunol., 6:485 (1988)). Included among its diverse biological activities is the induction of the terminal differentiation of B cells to plasma cells, the differentiation of killer T cells and nerve cells, as well as the acute phase protein synthesis of hepatocytes. It also stimulates the growth of hybridoma/plasmacytoma/myeloma cells, T cells, and hematopoietic stem cells. Differentiation inducing activity on megakaryocytes, leading to the production of platelets, has also been reported recently (Ishibashi, T., et al., Blood 74:1241, (1989)).
One example of glycosylated proteins or polypeptides possessing interleukin-6 activities is human interleukin-6 (hereafter referred to as hIL-6). There are a number of reports on processes for producing hIL-6; for example, production by human T cell hybridoma cells (Okada, M., et al., J. Exp. Med., 157:583 (1983)) or by human T cells transformed with human T cell leukemia virus (Japanese patent application, KOKAI NO. 61-115024). Human IL-6 may also be produced by recombinant DNA technologies which comprise transforming mammalian or bacterial cells with a vector carrying a DNA sequence encoding hIL-6 and then culturing these cells to obtain recombinant hIL-6. The resultant hIL-6 is a glycosylated protein if produced by mammalian cells, and an unglycosylated polypeptide if produced by bacterial cells. Both forms have been demonstrated to have interleukin-6 activities (EP 257406; WO 88/00206).
The mature fully functional hIL-6 polypeptide has 184 amino acid residues as predicted from the nucleotide sequence of its cDNA. However, polypeptides with one or more additional amino acid residues or (at most) 27 amino acid deletions at their N-terminus, as well as polypeptides with at most 50 amino acid deletions (or substitutions) at their C-terminus, are known to retain IL-6 activity (EP 257406; WO 88/00206; EP 363083; Brakenhoff, J. P. J., J. Immunol., 143:1175 (1989)).
Several methods have been used to try to prolong the plasma half-life of certain intravenously administered high molecular weight polypeptides. These include modification of the polypeptide with polyethylene glycol (PEG), dextran, poly[Glu-Lys], pullulan, modified polyaspartate or fatty acids, as well as coupling with gamma-globulin. The chemical modification with PEG of a few non-human derived enzymes, such as asparaginase, superoxide dismutase, or uricase, resulted in increased plasma half-life. However, a number of problems have been observed with PEGylation. Acylation of tyrosine residues on the protein can result in a lowering of the biological activity of the protein; certain PEG-protein conjugates are insufficiently stable and therefore find no pharmacological use; certain reagents used for PEGylation are insufficiently reactive and therefore require long reaction times during which protein denaturation and/or inactivation can occur. Also, the PEGylating agent may be insufficiently selective. Difficulties can also arise as a result of the hydrophobicity of the protein to be PEGylated; in an aqueous medium hydrophobic proteins resist PEGylation at physiological pH. The criteria for effective PEGylation include not only whether the conjugated molecule has a combination of increased serum half-life and decreased immunogenicity, but also whether it is in fact a more potent pharmacological agent than its unmodified parent molecule. Given the broad range of differences in the physical characteristics and pharmacokinetics among proteins, it is impossible to predict in advance whether a protein can be successfully PEGylated and/or whether the PEGylated protein will still retain its biological activity without inducing untoward immunological responses.
For example, in WO87/00056, relating to the solubilization of proteins for pharmaceutical compositions using polymer conjugation, the adverse effect of PEGylation on the in vitro activity of IL-2 is described in Example IIB (Table I, page 20). Example IC (page 19) references the IL-2 cell proliferation bioassay used. The results demonstrate that as more amino groups of the IL-2 are substituted with PEG, the PEGylated IL-2 undergoes a nearly 10-fold decrease in activity as compared to the activity of unmodified IL-2.
The covalent modification of lysine residues causes a reduction in bioactivity of certain proteins. Lysine modification with activated PEG-esters is random, difficult to control, and often results in reduced bioactivity of the modified protein. Goodson, R., et al., Bio/Technology, 8:343 (April 1990).
U.S. Pat. No. 4,904,584 (Feb. 27, 1990) describes a process for preparing PEGylated polypeptides. However, the process requires a premodification of the polypeptides by first preparing LDVs (lysine depleted variants) to obtain a polypeptide having a "suitable" number of reactive lysine residues. No evidence is presented that PEGylated derivatives were actually obtained; nor is their any evidence that these proposed modified polypeptides retained any biological activity. Further, there is no exemplification of the production of PEG-IL-6 nor any exemplification of retained activity.
As the in vivo half-life of IL-6 in blood is very short (Castell, J. V. et al., Eur. J. Biochem., 177:357 (1988)), it is desirable to increase hIL-6 plasma half-life and to thereby improve the pharmacokinetics and therapeutic efficacy of IL-6. To date, however, no one has been successful in so doing.